Polyketides have been produced in a variety of host cells, including Streptomyces, Saccharopolyspora, and Aspergillus for commercial purposes for many years. In particular, these compounds are often found in mycelial bacteria, the actinomycetes, in which the compounds are synthesized by enzymes known as polyketide synthases (PKSs) and produced as secondary metabolites. Typically, a polyketide was first identified as an active but uncharacterized ingredient in a soil or other environmental sample. Once an active ingredient was identified, then the organism that produced the ingredient was isolated and characterized. After the organism was characterized, it was often the subject of an intensive effort to increase the yield of the active ingredient. This effort typically involved successive rounds of subjecting the organism to mutagenic conditions, culturing the mutagenized organisms, and selecting those mutant organisms that produced the active ingredient in higher yields.
With the advent of molecular biology, the genes for the enzymes, called polyketide synthases or PKS(s), that perform the synthesis of certain polyketides became known. In some instances, such as, for example, the PKS enzymes that catalyze the synthesis of modular polyketides, the PKS enzymes are very large, multi-subunit proteins encoded by large gene clusters ranging from 10 kilobases (kb) to more than 100 kb in size. See, e.g., PCT patent publication No. 93/13663 (erythromycin); U.S. Pat. No. 5,098,837 (tylosin); U.S. Pat. No. 5,272,474 (avermectin); U.S. Pat. No. 5,744,350 (triol polyketide); and European patent publication No. 791,656 (platenolide), each of which is incorporated herein by reference. The cloning of these and other genes led to speculation that the yields of polyketide produced by these organisms could be increased by molecular biology techniques. See, e.g., U.S. Pat. No. 5,672,497. Despite these advances, however, there have been few, if any, reports of polyketide-producing strains that have been improved using such techniques.
Others working in the field developed novel methods and host cells not only for producing polyketides in host cells in which naturally occurring polyketide synthase genes had been eliminated or which otherwise did not produce polyketides but also for producing polyketides not otherwise found in nature in recombinant host cells. Thus, 6-deoxyerythronolide B has been produced in a Streptomyces coelicolor strain from which the endogenous actinorhodin gene cluster has been eliminated. See U.S. Pat. Nos. 5,672,491 and 5,712,146 and McDaniel et al., 1993, Engineered biosynthesis of novel polyketides, Science 262:1546-1550, each of which is incorporated herein by reference. In addition, the successful synthesis of a fungal polyketide, 6-methylsalicylic acid (6-MSA), has been reported in E. coli and in yeast. See Kealey et al., 1998, Production of a polyketide natural product in nonpolyketide producing prokaryotic and eukaryotic hosts, Proc. Natl. Acad. Sci. USA 95:505-509, and PCT patent publication No. 98/27203, incorporated herein by reference. Also, methods, reagents, and host cells were developed for producing novel polyketides from starting units not used by polyketide producing organisms in nature. See PCT patent publication No. 97/02358 and PCT patent application No. US98/14911. While the novel polyketides produced by such methods and cells were useful, yields were sometimes low, and rapid application of the technology in new host cells was sometimes hindered by endogenous recombination pathways and restriction--modification systems.
As one example, Kieser et al., December 1989, A mutation of Streptomyces lividans which prevents intraplasmid recombination has no effect on chromosomal recombination, Mol. Gen. Genet. 220(1): 60-64, reported on a recombination-deficient strain of S. lividans, JT46, originally characterized by Tsai and Chen, 1987, Isolation and characterization of Streptomyces lividans mutants deficient in intraplasmid recombination, Mol. Gen. Genet. 208: 211-218. This strain, however, produced the pigmented antibiotic actinorhodin produced by the unmodified parent strain S. lividans and so is not especially preferred for the production of other polyketides.
Other researchers have noted that actinorhodin as well as undecylprodigiosin and A-factor levels can be increased in Streptomyces lividans by transforming the strain with a vector having a copy number of 3-4 (but not 1-2) and encoding a phosphotyrosine protein phosphatase gene (ptpA) with its endogenous promoter isolated from S. coelicolor. See Umeyama et al., 1996, Expression of the Streptomyces coelicolor A3(2) ptpA gene encoding a phosphotyrosine protein phosphatase leads to overproduction of secondary metabolites in S. lividans, FEMS Microbiology Letters 144: 177-184; and Li and Strohl, January 1996, Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2), J. Bacteriology 178(1): 136-142. Unfortunately, actinorhodin has little if any therapeutic value.
There remains a need for host cells that can produce useful polyketides at higher levels than can be achieved with currently available cells, as well as a need for cells that can be transformed at high efficiency and can stably maintain extrachromosomal plasmids containing polyketide synthase genes over many generations. The present invention meets these and other needs.